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Primer, kit and method for detecting Dendroctonus pseudotsugae Hopkins

A technology of beetles and kits, applied in the field of molecular biology, can solve the problems of difficult identification, rapid identification, high personnel requirements, etc., and achieve good specificity

Inactive Publication Date: 2017-09-15
BEIJING FORESTRY UNIVERSITY
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  • Application Information

AI Technical Summary

Problems solved by technology

Among them, due to the complicated epidemic situation and the small size of adults (often less than 9mm), the rapid identification of bark beetles is very difficult. At present, less than 12% of the species can be identified at ports
[0003] At present, there are few researches on the Douglas beetle at home and abroad, and they mainly focus on the adult morphology. At the same time, the identification of the Douglas beetle at home and abroad is mainly through the adult morphological method, which requires professional knowledge of insect taxonomy. The requirements for personnel are high, and the douglas beetles carried in a large number of imported wood, wood products and wooden packages are complex, and it is difficult to deal with the study of adult morphology, and the port quarantine requires fast customs clearance, and it is impossible to raise eggs and larvae to re-identification after adult

Method used

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  • Primer, kit and method for detecting Dendroctonus pseudotsugae Hopkins
  • Primer, kit and method for detecting Dendroctonus pseudotsugae Hopkins
  • Primer, kit and method for detecting Dendroctonus pseudotsugae Hopkins

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Embodiment 1

[0046] The nested PCR detection method of embodiment 1 Huangshan beetle

[0047] (1) Reagents used: EZgene Insect gDNA Kit kit: purchased from BIOMIGA Company; SYBR PremixEx TaqⅡ (Perfect Real time): purchased from Bao Bioengineering (Dalian) Co., Ltd.; DL2000 DNA Marker: purchased from Bao Bioengineering (Dalian) Co., Ltd.; 1×Loading Buffer: purchased from Bao Biological Engineering (Dalian) Co., Ltd.

[0048] (2) DNA extraction: use the EZgene Insect gDNA Kit kit from BIOMIGA Company to extract target insects (including Beetles pine-toothed beetles, Beetles largii, Beetles largii, Beetles punctatus and Beetles pine). ) gDNA, dilute the DNA to 50ng / μL.

[0049] (3) Using gDNA as a template, the first round of PCR amplification of the COI sequence was performed using universal primers (Table 1-1), with a fragment size of 700 bp.

[0050] Table 1 Sequences of general primers LCO1490 and HCO2198

[0051]

[0052] The PCR reaction system is shown in Table 2.

[0053] Table...

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Abstract

The invention provides a primer, a kit and a method for detecting Dendroctonus pseudotsugae Hopkins. The detection method is nested PCR, and the primer used in the invention is represented by SEQ ID NO.1 and SEQ ID NO.2. The invention also provides a Dendroctonus pseudotsugae Hopkins detection kit containing the primer. The detection method and the kit have the advantages of simplicity in operation, good accuracy and reliability, short detection time and good specificity, and can distinguish Dendroctonus pseudotsugae Hopkins and other bark beetles, and the detection limit is 20 pg.

Description

technical field [0001] The invention relates to the field of molecular biology, and more specifically, to primers, a kit and a detection method for detecting the douglas beetle. Background technique [0002] Dendroctonus pseudotsugae Hopkins. is the most frequently intercepted beetle at ports in recent years, and it seriously endangers forest and timber resources. In recent years, the wood pests carried in a large number of imported wood, wood products and wooden packaging are extremely complex, and the common ones are longhorn beetles, bark beetles, long beetles, gill beetles, and weevils. Among them, bark beetle is very difficult to quickly identify because of its complicated epidemic situation and small adults (often less than 9mm). At present, less than 12% of the species can be identified at ports. According to statistics, the interception frequency of the Huangshan beetle accounts for 36.31% of the total interception frequency of the beetle, which is 29.3% higher than...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6888
Inventor 石娟邬颖陈芳王子楠李建光
Owner BEIJING FORESTRY UNIVERSITY
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